|
Streak a fresh plate of cells.
- Make LB plates.
- Make LB media.
- Make minimal plates with arginine, use extra amino acids as appropriate for strain K.
- Make minimal plates without arginine, use extra amino acids as appropriate for strain K.
- Make LB plates with ampicillin (2000 mg/mL) or tetracycline (200 mg/mL).
- Make minimal media, except no phosphates and add 0.05 M Tris and 0.05 M maleic acid (TM buffer). Adjust the pH to 6.0.
- Make 0.50 mg/mL NTG solution (10 mg in 20 mL sterile H2O).
- Make 1.0 mg/mL NTG solution (20 mg in 20 mL sterile H2O).
- Make minimal media (M9).
Make an ON in LB (10 mL), incubate at 37 ¼C with shaking.
Dilute the ON 50-fold in LB (0.2 mL in 10 mL) and incubate a few hours, until the culture is turbid, but only about 10-20% the level of the ON.
Remove a 1 mL aliquot for control plating. Plate on antibiotic + and - plates.
Remove two 10 mL aliquots of the culture (sample A and sample B).
- Spin each culture (samples A and B) at 5K. Discard super and resuspend in 10 mL TM-buffer.
- Spin each culture at 5K. Discard super and resuspend in 10 mL TM-buffer.
- Spin each culture at 5K. Discard super and resuspend in 10 mL TM-buffer.
Remove 1 mL aliquot from each culture (9 mL remain) for control plating. Plate on antibiotic + and - plates.
Add 1 mL of 0.50 mg/mL NTG solution to the washed cells of sample A. Makes 10 mL total volume and a 50 mg/mL final concentration of NTG.
Incubate at 37 °C for 20 min.
Add 1 mL of 1.00 mg/mL NTG solution to the washed cells of sample B. Makes 10 mL total volume and a 100 mg/mL final concentration of NTG.
Incubate at 37 °C for 20 min.
Remove 1 mL aliquot from each culture for control plating. Plate on antibiotic + and - plates.
- Pellet cells (5K) and resuspend in 10 mL cold minimal media.
- Pellet cells (5K) and resuspend in 10 mL cold minimal media.
- Pellet cells (5K) and resuspend in 10 mL LB media.
Grow the cells at 37 °C, with shaking for about 3-4 hours or overnight.
- Make dilutions of the cells (10-2 - 10-8) in minimal media.
- Spread 0.05 mL of the 10-2 dilution onto an LB plate. Do this in duplicate.
- Spread 0.05 mL of the 10-4 dilution onto an LB plate. Do this in duplicate.
- Spread 0.05 mL of the 10-6 dilution onto an LB plate. Do this in duplicate.
- Spread 0.05 mL of the 10-8 dilution onto an LB plate. Do this in duplicate.
- (Spread the cell solution with a glass rod until the surface of the plate is dry.)
- Save dilutions to re-plate.
- Incubate plates ON at 37 °C. The goal is a plate with about 100 colonies per plate.
From the dilution that gives around 100 colonies per plate, make 50 more plates (spread 0.05 mL of the dilution onto an LB plate). Incubate plates ON at 37 °C. The goal is a plate with about 100 colonies per plate.
Replica plate the 50 plates with 100 colonies each onto arg- plates, arg+ plates and LB plates. Be sure to press the arg- plate first after pressing the plate of colonies. Incubate over night at 37 °C.
Superimpose the arg- plate over the arg+ plate and see if any colonies are missing from the minus plate. If so, pick the colony from the arg+ plate and streak it out on an LB plate to get many well-isolated colonies. Incubate ON at 37 °C.
Pick several isolated colonies and streak each one on an arg- plate and an arg+ plate. Streak a smear of cells onto the arg- plate, and then without flaming the loop, streak a smear onto the arg+ plate. Flame the loop and streak each plate for single colonies. Incubate ON at 37 °C.
Auxotrophs will only grow on the arg+ plates. Pick a colony from the arg+ plate and streak it out on an LB plate to get many well-isolated colonies. Incubate ON at 37 °C.
Pick several isolated colonies and streak each one on an arg- plate and, then an arg+ plate. Incubate ON at 37 °C.
Make an ON of an arg auxotroph.
Titer Pf1 on the arg auxotrophic cells, to test whether or not Pf1 can use the mutant cell line as a host.
Record the titer results.
back to top
|
